Use of a Pollen-Based Diet to Expose the Ladybird Beetle Propylea japonica to Insecticidal Proteins

A rape seed pollen-based diet was developed and found to be suitable for use in a dietary exposure assay for Propylea japonica. Using the diet, we established and validated a dietary exposure assay by using the protease inhibitor E-64 as positive control. Dose-dependent responses were documented for all observed life-table parameters of P. japonica including survival, pupation and eclosion rates, development time and adult weight. Results suggested that the dietary assay can detect the effects of insecticidal compounds on the survival and development of P. japonica. Using the established dietary assay, we subsequently tested the toxicity of Cry1Ab, Cry1Ac and Cry1F proteins that are expressed by transgenic maize, cotton or rice plants to P. japonica larvae. The diet containing E-64 was included as a positive control. Survival and development of P. japonica larvae were not adversely affected when the diet contained purified Cry1Ab, Cry1Ac, or Cry1F at 500 µg/g diet representing a worst-case exposure scenario. In contrast, P. japonica larvae were adversely affected when the diet contained E-64. The bioactivity and stability of the Cry proteins in the diet and Cry protein uptake by the ladybird larvae were confirmed by bioassay with a Cry-sensitive insect species and by ELISA. The current study describes a suitable experimental system for assessing the potential effects of gut-active insecticidal compounds on ladybird beetle larvae. The experiments with the Cry proteins demonstrate that P. japonica larvae are not sensitive to Cry1Ab, Cry1Ac and Cry1F.     

Exploring the main factors influencing habitat preference of Popillia japonica in an area of recent introduction

The possibility of establishment of an invasive species in an area of new introduction is influenced by several factors, including weather conditions, soil characteristics, and species spread dynamics. Disentangling the role of multiple variables on habitat preference can provide important elements supporting the management of invasive species. In this work, we present a modelling framework that allows i) the identification of the main drivers influencing the probability of the presence of the invasive pest Japanese beetle Popillia japonica, in an area of recent introduction in Northern Italy, and ii) the evaluation of bi-dimensional niches to investigate the non-linear combined effects of couples of variables on the potential distribution of this species. Occurrence records of larval populations sampled between 2017 and 2019 in 8488 sites in Northern Italy were used in the present study. The sample sites were characterised based on a set of explanatory variables related to land use, soil characteristics, meteorological conditions, and infestation history. We performed a stepwise logistic regression analysis for testing the statistical significance of explanatory variables on the occurrence of local larval populations. Then, we implemented a Generalized Additive Model to investigate the bi-dimensional ecological niche determined by the interaction of two variables on the probability of the presence of P. japonica. The results highlight the prominent role of soil and weather variables and the infestation history of the species in driving the presence of P. japonica larvae. Less acidic soils, especially with sandy-skeletal particles, are preferred. A high density of P. japonica larvae is associated with medium content of soil organic carbon in the soil. Long drought periods or high precipitation levels reduce the probability of the presence of the species. Non-linear effects are observed between soil temperature variables and habitat preferences of P. japonica. The probability of the presence of P. japonica is also related to the number of years since the first occurrence of the species at the site. The results of the present work provide fundamental elements for guiding the assessment of the risk and the management of P. japonica.     

Intranuclear disease of uncertain etiology in larvae of the blackfly,Simulium vittatum

An intranuclear disease of uncertain etiology has been found in larvae of the blackfly, Simulium vittatum (Diptera: Simuliidae). The disease, which causes little mortality, is characterized by a lack of pigmentation in the cuticle and the occurrence of melanotic deposits in the cytoplasm of fat body and midgut epithelial cells. A single, dense lacunate inclusion develops within the nuclei of these cells, ranging from 20 to 30 μm in midgut epithelial cells, and averaging 18 μm in fat body cells. The disease has been reproduced in the laboratory by feeding first-instar blackfly larvae on homogenates of diseased larvae. However, attempts to isolate a causative agent have not been successful. The cytopathology of the disease resembles the cytopathology caused by densonucleosis viruses, but particles, either free or in aggregations, typical of the virions of this virus type have not been identified in infected nuclei.     

Comparative histopathology of infection byOvavesicula popilliae [Microsporida: Pleistophoridae] in larval and adult Japanese beetles,Popillia japonica

The host response to infection and tissue susceptibility of larval and adult Japanese beetles,Popillia japonica Newman, to the microsporidium,Ovavesicula popilliae Andreadis & Hanula, are reported. The normally transparent Malpighian tubules of Japanese beetle larvae, were hypertrophied and white in color when infected withO. popilliae, a microsporidian which also infects larval fat body, epidermis and pericardial cells. In addition to these tissues, œnocytes and tracheal epithelial cells were also infected in adults. Adult and larval reactions to infection included hypertrophied cells and melanization of the pericardium, but only larvae exhibited an intense inflammatory response. The discoloration of the pericardium most likely resulted from an accumulation of melanin.      

Effectiveness of Steinernema spp. and Heterorhabditis bacteriophora against Popillia japonica in the Azores

Steinernema carpocapsae (Breton strain), S. glaseri, and Heterorhabditis bacteriophora were evaluated for their potential to control immature stages of the Japanese beetle, Popillia japonica, on Terceira Island (the Azores). In bioassays carried out at temperatures higher than 15 C, S. glaseri and H. bacteriophora caused 100% mortality of larvae, whereas S. carpocapsae caused 56% larval mortality. At temperatures slightly below 15 C, only S. glaseri remained effective. In field plots, in September, S. glaseri and S. carpocapsae reduced larval populations by 91% and 44%, respectively, when applied at the rate of 10⁶ nematodes/m². In April, S. glaseri caused 31% reduction in numbers of larvae, but S. carpocapsae was ineffective. In colder months (November-February) neither steinernematids nor H. bacteriophora reduced larval populations. Increasing the application rate from 10⁶ to 5 x 10⁶ infective stage S. glaseri per m² increased efficacy from 63% to 79% mortality.     

Reovirus-like particles and their vertical transmission in the Mexican bean beetle,Epilachna varivestis (Coleoptera: Coccinellidae)

Examination of thin sections of organs and tissues from the Mexican bean beetle, Epilachna varivestis, adults and larvae revealed the consistent presence of isometric, reovirus-like particles in the cytoplasm and rarely in the nuclei. Cytoplasmic inclusions having a dense and finely granular matrix with virus-like particles at their periphery were noticed occasionally. These inclusions were, however, very frequent in the nurse cells in the germarium of the ovariole. Virus-like particles were found in the cytoplasm of oocytes and eggs as well as embedded in the nuclei of sperm, thus suggesting vertical transmission of these particles, and explaining the 100% infection of the Mexican bean beetle colony maintained at the Virology and Biocontrol Laboratory of the University of Arkansas. Partially purified extracts from ovarioles or entire beetles contained particles which, in negatively stained preparations, resembled reoviruses without the external protein coat. It is suggested that these particles represent a latent virus of the Mexican bean beetle.     

Control of the bacterium Serratia marcescens in an insect host-parasite rearing program

Two occluded viruses of the Entomopoxvirus (D)/¹: ¹/¹: $\text{X}\text{X}\text{:}\text{I}\text{O}$ (= Vagoiavirus) group have been found in larvae of Dermolepida albohirtum (Scarabaeidae: Melolonthinae) and Aphodius tasmaniae (Scarabaeidae: Aphodiinae) from northern Queensland and northern Tasmania, Australia, respectively. Electron microscopical studies have been made of thin sections of occluded (mature) and nonoccluded virus particles within the fat body tissue of living diseased D. albohirtum larvae and of occluded virus particles within a dead, field-collected A. tasmaniae larva. The morphology and development of the known Australian entomopoxviruses are compared with previously known entompox or spheroidosis viruses from various insects.     

Pathology and Development of the Grasshopper Inclusion Body Virus in Melanoplus sanguinipes

Grasshoppers, Melanoplus sanguinipes (F.), infected with the grasshopper inclusion body virus (GIBV) showed a general torpor, took longer to develop, and had abnormally high rates of mortality. Infection was found only in the fat body, and developing viruses and inclusion bodies were observed in the nuclei and cytoplasm of infected cells. Although the size of the inclusion bodies in cells varied at different stages of infection, the inclusion bodies appeared to grow during the infection. Electron microscopic investigations of viral replication showed that at about 8 days after inoculation presumptive viral particles had developed as buds or protrusions from precursor granular masses; thereafter, these particles underwent internal differentiation and were incorporated into developing inclusion bodies. The GIBV was similar to insect viruses in the genus Vagoiavirus Weiser and to pox viruses, particularly vaccinia.     

捕食者对空心莲子草叶甲种群的生物胁迫

广食性捕食者广泛捕食植食性昆虫,常被用于有害生物的生物防治,也因此影响植食性昆虫对杂草的生物效果。空心莲子草叶甲(Agasicles hygrophila)(鞘翅目:叶甲科Chrysomelidae)作为入侵恶性杂草空心莲子草(Alternanthera philoxeroides)(苋科:莲子草属Alternanthera)的专性天敌,从美国的弗罗里达州引入中国,在释放地防治空心莲子草取得了较好的防治效果。虽然空心莲子草叶甲在引入地均已建立田间种群并有一定程度的自然扩散,但丰富的食物资源,并未使空心莲子草叶甲的自然种群数量变得繁荣,因此其未能有效抑制空心莲子草的扩散蔓延。在野外调查时发现空心莲子草生境中存在大量广食性捕食者。这些广食性捕食者是抑制空心莲子草叶甲种群数量扩张的生物胁迫因子吗?为此,选择捕食性昆虫龟纹瓢虫(Propylaea japonica)(鞘翅目:瓢虫科Coccinellidae)、蜘蛛类捕食者拟水狼蛛(Pirata subpiraticus)(蜘蛛目:狼蛛科Lycosidae)与斜纹猫蛛(Oxyopes sertatus)(蜘蛛目:猫蛛科Oxyopidae)为捕食者,分别以空心莲子草叶甲各虫态为猎物,构建简单的捕食者-猎物系统,在室内检测了上述3种捕食者对空心莲子草叶甲各虫态在不同密度下的日捕食量,以期了解捕食者对空心莲子草叶甲的捕食作用,客观评估空心莲子草叶甲的生物防治效能。研究结果表明:捕食者龟纹瓢虫、斜纹猫蛛与拟水狼蛛均捕食空心莲子草叶甲的卵粒及1龄、2龄幼虫,斜纹猫蛛与拟水狼蛛捕食3龄幼虫,捕食者的捕食量均随着猎物密度的升高而增加,寻找效应降低。三者均不捕食成虫。除拟水狼蛛对3龄幼虫的捕食用Holling II模型拟合不呈显著相关关系外,其余捕食反应均拟合Holling II模型并显著相关。通过拟合方程得出捕食者对空心莲子草叶甲卵粒的理论日最大捕食量为:斜纹猫蛛10.9粒,拟水狼蛛为6.2粒,龟纹瓢虫为5.6粒;对1龄幼虫的理论日最大捕食量为:斜纹猫蛛为17.1头;拟水狼蛛为35.8头,龟纹瓢虫为10.4头;对2龄幼虫的理论日最大捕食量为:斜纹猫蛛为6.6头,拟水狼蛛为11.2头,龟纹瓢虫为2.9头;对3龄幼虫的理论日最大捕食量为:斜纹猫蛛捕食12.3头,拟水狼蛛为1.1头。研究结果证实了捕食者可通过捕食作用降低空心莲子草叶甲种群密度,削弱空心莲子草叶甲对空心莲子草的控害效能,是空心莲子草叶甲种群存活的生物胁迫因子。建议在提高空心莲子草叶甲田间种群数量,达到对空心莲子有效的持续控制效果方面开展进一步研究。     

Developmental stages and gut microenvironments influence gut microbiota dynamics in the invasive beetle Popillia japonica Newman (Coleoptera: Scarabaeidae)

Popillia japonica Newman (Coleoptera: Scarabaeidae) is a highly polyphagous invasive beetle originating from Japan. This insect is highly resilient and able to rapidly adapt to new vegetation. Insect-associated microorganisms can play important roles in insect physiology, helping their hosts to adapt to changing conditions and potentially contributing to an insect's invasive potential. Such symbiotic bacteria can be part of a core microbiota that is stably transmitted throughout the host's life cycle or selectively recruited from the environment at each developmental stage. The aim of this study was to investigate the origin, stability and turnover of the bacterial communities associated with an invasive population of P. japonica from Italy. Our results demonstrate that soil microbes represent an important source of gut bacteria for P. japonica larvae, but as the insect develops, its gut microbiota richness and diversity decreased substantially, paralleled by changes in community composition. Notably, only 16.75% of the soil bacteria present in larvae are maintained until the adult stage. We further identified the micro-environments of different gut sections as an important factor shaping microbiota composition in this species, likely due to differences in pH, oxygen availability and redox potential. In addition, P. japonica also harboured a stable bacterial community across all developmental stages, consisting of taxa well known for the degradation of plant material, namely the families Ruminococcacae, Christensenellaceae and Lachnospiraceae. Interestingly, the family Christensenallaceae had so far been observed exclusively in humans. However, the Christensenellaceae operational taxonomic units found in P. japonica belong to different taxonomic clades within this family.     

Histopathology of virus-like particles in Heliothis spp

Larvae of Heliothis spp. collected from cotton, soybean, and peanut fields in South Carolina were found to be infected with virus-like particles (VLPs). Infected larvae became pale and swollen, stopped feeding, and remained alive for 2–3 weeks. Hemolymph from these larvae was milky and contained numerous spherical bodies ranging in diameter from 2 to 10 μm. The hemolymph also contained VLPs which were oval and measured 375 × 125 nm. Infectivity tests with crude saline extracts of infected larvae demonstrated that the pathogen could be transmitted by injection but not per os. The spherical bodies contained VLPs (387 × 149 nm) surrounded by two envelopes and packed together in clusters. These VLPs were also found in fat body cells, cuticular epithelial cells, tracheal cells, and connective tissue associated with the body wall and the gut. They were not found in muscle tissue or in midgut epithelial cells. Similar VLPs have been found in Heliothis zea from Mississippi and Trichoplusia ni from California, but a positive identification of the VLPs has not been made in any of these studies. Morphologically they appear to be distinct from any other previously described insect viruses.     

Synthesis and release of proteins from cultured larval fat body of the southwestern corn borer,Diatraea grandiosella

The larval fat body of the southwestern corn borer, Diatraea grandiosella, was cultured in vitro to examine the relationship between proteins present in the fat body, those released into the medium, and those present in the haemolymph. While the incorporation of [³H]leucine into fat body proteins was high in last instar pre-diapausing and non-diapausing larvae, it fell in early diapausing larvae to about 11% of that found in prediapausing larvae. Incorporation of [³H]leucine into the diapause-associated protein of the fat body increased gradually in pre-diapausing larvae and reached a maximum in newly-diapaused larvae at a time when the incorporation of [³H]leucine into other proteins of the fat body had declined. The proteins released from the cultured fat body showed identical electrophoretic properties and close immunochemical relationships to most of those present in the haemolymph. Small amounts of the diapause-associated protein were released in vitro from the fat body of larvae of different ages in diapause. Lipophorin was also released in vitro from the fat body of non-diapausing and diapausing larvae, and shown to be immunochemically identical to the lipophorin present in the haemolymph.     

Hormonal regulation of proline synthesis and glucose release in the fat body of the Colorado potato beetle, Leptinotarsa decemlineata

In the adult males of the Colorado potato beetle, Leptinotarsa decemlineata, corpora cardiaca extract injected in vivo gives rise to an increase in glucose and a decrease in alanine concentration of the haemolymph. The regulation of proline synthesis and glucose release in the fat body of the Colorado potato beetle was investigated further in vitro. Proline regulates its own synthesis by a feedback inhibition. Moreover, a factor present in extracts from the corpora cardiaca stimulates synthesis in the fat body in vitro. This effect was demonstrated with corpora cardiaca extracts from beetles, locusts and cokroaches. Also, synthetic adipokinetic hormone stimulates proline synthesis in the fat body of the Colorado beetle. In addition, a release of glucose from the fat body of the beetle could be evoked by the addition of locust and beetle corpora cardiaca extracts or synthetic adipokinetic hormone. The physiological significance of both effects is discussed.     

Differential susceptibility of Popillia japonica 3rd instars to Heterorhabditis bacteriophora (Italian strain) at three different seasons

The scarab beetle Popillia japonica, a pest native to northern Japan, has been recently found in Italy. Entomopathogenic nematodes are useful for biological control of this invasive insect. Previous work showed that 1st and 2nd larval stages are more susceptible to nematodes than 3rd instars. We tested the effectiveness of Heterorhabditis bacteriophora in the laboratory against P. japonica 3rd instars. Experiments were conducted in Italy with larvae field collected in the fall, winter and spring, showing a significant decrease in effectiveness from the fall to spring.     

Ultrastructure and respiration of the fat body of diapausing and non-diapausing larvae of the corn borer,Diatraea grandiosella

A comparative study of the fat body of diapausing and non-diapausing larvae of the corn borer, Diatraea grandiosella, was undertaken using the electron microscope and the oxygen electrode. The electron microscopic results showed a shift from a synthetic to a storage function taking place in a 1 to 2 day period during the final instar of non-diapausing larvae, and in a 4 to 8 day period in that of pre-diapausing larvae. This transition was characterized by a decrease in the number of mitochondria and amount of rough endoplasmic reticulum, and by an increase in the number of proteinaceous granules and lysosomes. In vitro measurements using the oxygen electrode showed that the fat body is a normal aerobic respiratory tissue. The tissue reacted in a predictable manner to inhibitors of oxidative metabolism, including malonate, rotenone, oligomycin, and antimycin, and to the uncoupler, dinitrophenol. During the last instar the observed decrease in the respiratory rate of the fat body coincided with the observed ultrastructural changes in its cells. The fat body of 75 day old environmentally induced and juvenile hormone induced diapausing larvae consumed 90% and 78% less oxygen, respectively than that of 14 day old non-diapausing larvae.     

<Emphasis Type="Italic">Hexamermis popilliae</Emphasis> n. sp. (Nematoda: Mermithidae) parasitizing the Japanese beetle <Emphasis Type="Italic">Popillia japonica</Emphasis> Newman (Coleoptera: Scarabaeidae) in Italy

A new species of mermithid nematode, Hexamermis popilliae n. sp. (Nematoda: Mermithidae) is described from the Japanese beetle Popillia japonica Newman in Italy, an area of new introduction for this invasive pest. The combination of the following characters separates H. popilliae from other members of the genus Hexamermis Steiner, 1924: adult head obtuse; amphidial pouches slightly posterior to lateral head papillae in female but adjacent to lateral head papillae in males; amphidial openings large, well developed; amphidial pouches elliptical in females and oblong in males; cuticular vulvar cone well developed, vulvar lips greatly reduced or lacking, vagina curved at tip where meeting uteri, without reverse bend (not S-shaped), spicules slightly curved, with a slight bend in the basal portion, approximately equal to body width at cloaca. This is the first record of a species of Hexamermis parasitizing the Japanese beetle Popillia japonica. The only previous mention of mermithid nematodes from P. japonica was an undescribed species of Psammomermis in North America. Hexamermis popilliae will be evaluated as a potential biological control agent in an integrated control program of the Japanese beetle in Italy.     

Uptake of beta-ecdysone by the fat body and membrane vesicles of fat body cells of Sarcophaga peregrina larvae.

The fat body of Sarcophaga peregrina larvae was shown to incorporate ³H-β-ecdysone when it was incubated with the hormone in vitro. Most of the incorporated radioactivity was found in the cytoplasmic fraction as free β-ecdysone, not as a protein-β-ecdysone complex.Rapid uptake and accumulation of β-ecdysone was observed in the membrane vesicles of fat body cells in vitro. The apparent K_m value for uptake was estimated to be 1·25 × 10^?7 M. The β-ecdysone in the membrane vesicles was rapidly released when 2,4-dinitrophenol was added. These results suggest that β-ecdysone was incorporated into the membrane vesicles by active transport and not by free diffusion. The hormone is probably incorporated into larval tissues by the same mechanism as it is incorporated into the membrane vesicles of fat body cells.     

Geographic variation in phenotypic traits in Phratora spp. and the effects of conditioning on feeding preference

The leaf beetle genus Phratora (L.) (Coleoptera: Chrysomelidae) includes important pests of cultivated willows and poplars. The feeding preference of P. vulgatissima and P. vitellinae collected from different geographical locations was studied under laboratory conditions. There were geographic differences in the amount consumed of different willow host genotypes by each beetle species. Correlation analysis showed that, regardless of these individual differences between willow genotypes and locations, the ranking of preferred host genotypes was significantly related between locations. There were differences in the preference of host genotypes by P. vulgatissima adults and larvae after being confined to a specific willow genotype for a month, suggesting the possibility of a conditioning effect on food preference. This study also permitted the assessment of possible geographic differentiation in morphological traits. Both beetle species generally showed an increase in body size, width, and percentage fat of dry body weight with a more northerly location. These results are discussed in relation to using varietal mixtures as a pest management strategy.     

An immunochemical study of the diapause-associated protein of the southwestern corn borer,Diatraea grandiosella

The distribution and titre of the diapause-associated protein (DAP), which accumulates in the fat body of diapausing larvae of the southwestern corn borer, Diatraea grandiosella, were determined using an antibody raised to the purified protein. Immunoblotting and immunodiffusion showed that the highest concentration of DAP was present in the fat body. Small amounts of the protein were present in the haemolymph and even lower levels were detected in other tissues. Rocket immunoelectrophoresis showed that DAP accumulated in the fat body at the beginning of diapause, reached a plateau, and gradually declined towards the end of diapause. Similarly, the titre of DAP in last instar non-diapausing larvae treated with a JH analogue increased, reached a plateau and then declined. Double immunodiffusion and immunoblotting using the DAP-antibody and extracts of the fat body of D. grandiosella and the southern cornstalk borer, Diatraea crambidoides, and the sugarcane borer, Diatraea saccharalis, revealed the partial immunochemical identity of the DAP of D. grandiosella and a protein present in these two species. Denaturing electrophoresis with immunoblotting showed that the DAP-related proteins of D. crambidoides and D. saccharalis have apparent molecular weights of 33,000 and 36,000, respectively, as compared to a molecular weight of 35,000 for DAP of D. grandiosella. A dot blot analysis showed that no cross reaction occurred between the “diapause proteins” present in the haemolymph of adults of the Colorado potato beetle, Leptinotarsa decemlineata, and the DAP of D. grandiosella.     

Protein metabolism by the salivary glands and other organs of the larva of the blowfly, Calliphora erythrocephala

Protein metabolism in salivary glands, gut, haemolymph, and fat body during the last larval instar of the blowfly, Calliphora erythrocephala, has been investigated. In salivary glands, protein release, protein synthesis, amylase, and pepsin-like protease activity were maximal in 6 day larvae, this being at a time when the larvae had finished feeding. All these functions declined in glands from the rounded-off white puparial stage (R.O.) while acid phosphatase activity rose throughout the third instar to a maximum at the R.O. stage, Glands from 6 and 7 day larvae released protein which on disk gel electrophoresis separated into four minor bands and two major bands one of the latter possessing protease activity.In the gut, pepsin-like protease activity was maximal in 4 day larvae after which it fell rapidly thus following the feeding pattern of the larva in contrast to that in the salivary glands which did not.In vitro experiments showed that protease was released from 6 day glands through the basal membrane of the cells and not via the duct. A pepsin-like protease was also found in the haemolymph and fat body, the activity in the fat body rising rapidly during the latter part of the third instar, a rise which is attributed to the fat body sequestering protease from the haemolymph. Acid phosphatase activity in the fat body was maximal in 5 day larvae indicating that this enzyme was synthesized early in the third instar. It was shown that fat body sequestered ¹⁴C-labelled protein synthesized by and released from the salivary glands, most of the ¹⁴C activity being associated with a 600 g precipitable, acid-phosphatase rich fraction.It is proposed that in late third instar larvae the salivary glands function as glands of internal secretion, releasing protease into the haemolymph, which is then sequestered by the fat body (and perhaps other tissues) and is subsequently used in the lysis of the tissues at the time of metamorphosis.